the pd-l1/pd-1 axis blocks neutrophil cytotoxicity in cancer · 28/02/2020 · study (granot et...

THE PD-L1/PD-1 AXIS BLOCKS NEUTROPHIL CYTOTOXICITY IN CANCER

Maya Gershkovitz1#, Olga Yajuk1#, Tanya Fainsod-Levi1 and Zvi Granot1*.

1 Department of Developmental Biology and Cancer Research, Institute for Medical Research

Israel Canada, Hebrew University Medical School, 91120, Jerusalem, Israel.

# Contributed equally

* Corresponding author – Zvi Granot, Department of Developmental Biology and Cancer

Research, Institute for Medical Research Israel Canada, Hebrew University Medical School,

91120, Jerusalem, Israel. [email protected]

ABSTRACT

The PD-L1/PD-1 axis was shown to promote tumor growth and progression via the inhibition

of anti-tumor immunity. Blocking this axis was shown to be beneficial in maintaining the anti-

tumor functions of the adaptive immune system. Still, the consequences of blocking the PD-

L1/PD-1 axis on innate immune responses remain largely unexplored. In this context, neutrophils

were shown to consist of different subpopulations, which possess either pro- and anti-tumor

properties. PD-L1-expressing neutrophils are considered pro-tumor as they can suppress

cytotoxic T-cells. That said, we found that PD-L1 expression is not limited to tumor promoting

neutrophils but is also evident in anti-tumor neutrophils. We show that neutrophil cytotoxicity is

effectively and efficiently blocked by tumor cell expressed PD-1. Furthermore blocking of either

neutrophil PD-L1 or tumor cell PD-1 maintains neutrophil cytotoxicity resulting in enhanced

tumor cell apoptosis. Importantly, we show that tumor cell PD-1 blocks neutrophil cytotoxicity

and promotes tumor growth via a mechanism independent of adaptive immunity. Taken together,

these findings highlight the therapeutic potential of enhancing anti-tumor innate immune

responses via blocking of the PD-L1/PD-1 axis.

.CC-BY-NC-ND 4.0 International licenseavailable under a(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprintthis version posted February 29, 2020. ; https://doi.org/10.1101/2020.02.28.969410doi: bioRxiv preprint

https://doi.org/10.1101/2020.02.28.969410

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INTRODUCTION

Anti-tumor immune surveillance is an important feature of the immune system where immune

cells recognize and eliminate malignancies (Chen and Mellman, 2013; Hanahan and Coussens,

2012; Nanda and Sercarz, 1995). However, immune cells make a significant proportion of the

tumor microenvironment (TME) and they were shown to contribute to tumor development and

progression (Barberis et al., 2016). This dramatic change in immune function is mediated via

various evasion mechanisms (Drake et al., 2006; Ribas, 2015), one of which is the expression of

immune checkpoint molecules by tumor cells. Under physiological conditions, the expression of

immune checkpoint molecules regulates immune responses and prevents autoimmunity (Sharma

and Allison, 2015; Topalian et al., 2015). In cancer, the expression of these molecules enables

blocking of anti-tumor immunity and promotes immune evasion (Ribas, 2015).

The programmed cell death protein 1 (PD-1; also known as Pdcd1) receptor and its ligand

PD-1 ligand 1 (PD-L1) serve as an immune checkpoint pathway and are of significant clinical

importance (Chikuma, 2016; Trivedi et al., 2015). Tumor cells benefit from the expression of

PD-L1 as its expression inhibits the proliferation and activation of cytotoxic T cells (Amarnath et

al., 2011; Blank et al., 2004; Butte et al., 2007). Interestingly, the expression of the inhibitory

ligand, PD-L1, is not limited only to tumor cells in the TME (Gibbons Johnson and Dong, 2017).

Neutrophils in the TME and in the peritumoral tissue also express high levels of PD-L1 (He et

al., 2015; Wang et al., 2017). Tumor-infiltrating neutrophils were shown to play important, yet

multifaceted and sometimes opposing roles (Sionov et al., 2015b). The tumor promoting

properties of neutrophils were widely described as they have the ability to initiate tumorigenesis

(Katoh et al., 2013), enhance tumor progression via direct stimulation of tumor cells proliferation

(Houghton et al., 2010), enhance angiogenesis (Jablonska et al., 2010; Nozawa et al., 2006),

suppress anti-tumor immune effector cells (de Kleijn et al., 2013; Gabrilovich et al., 2012) and

promote metastatic spread (Casbon et al., 2015; Cools-Lartigue et al., 2013; Spicer et al., 2012).

In contrast, studies have shown that neutrophils may also possess anti-tumor features as they can

kill tumor cells via the secretion of H2O2 (Gershkovitz et al., 2018a; Granot et al., 2011). The

role of PD-L1pos neutrophils is associated with a tumor-promoting phenotype, they were found to

suppress cytotoxic T cells, thereby enhance disease progression and limit patient survival (de

Kleijn et al., 2013; He et al., 2015).



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Although the expression of PD-L1 is mainly attributed to tumor cells and PD-1 expression is

attributed to T cells, the expression pattern of PD-L1/PD-1 in cancer is more complex (Gibbons

Johnson and Dong, 2017; Simon and Labarriere, 2017). A study by Kleffel and colleges (Kleffel

et al., 2015) showed that murine and human melanomas contain PD-1 expressing cancer

subpopulations and demonstrate that melanoma PD-1 cell-intrinsic signaling promotes

tumorigenesis. Although the role of PD-L1 on neutrophils is not fully understood, this

observation raises the possibility that PD-L1pos neutrophils can directly interact with PD-1pos

tumor cells. In this study, we examined the role played by the PD-L1/PD-1 axis in regulating

neutrophil cytotoxicity toward tumor cells. We found that neutrophil PD-L1 not only acts to

suppress T cell responses, but can also directly inhibit neutrophil cytotoxicity. Blocking the PD-

L1/PD-1 interaction enhances tumor cells susceptibility to neutrophil cytotoxicity. In a previous

study (Granot et al., 2011), we showed that neutrophils possess an anti-metastatic role when

encountering tumor cells in the pre-metastatic niche. In line with these observations, we found

that PD-1 levels in tumor cells have a significant impact on the anti-metastatic function of

neutrophils. While neutrophils efficiently eliminate control tumor cells, PD-1 over-expressing

tumor cells are resistant to neutrophil cytotoxicity. Our observations provide a wider scope to the

role PD-L1 plays on neutrophils in cancer, and highlight the therapeutic potential of blocking the

PD-L1/PD-1 axis not only in propagating anti tumor T cell responses but also in facilitating

neutrophil cytotoxicity.

RESULTS

Immature low-density neutrophils express higher levels of PD-L1

The PD-L1/PD-1 immune checkpoint is well characterized in the context of cancer where PD-

L1-expressing tumor cells were shown to functionally inhibit the cytotoxicity of PD-1-

expressing T cells. Furthermore, blocking of PD-L1/PD-1 interaction results in the maintenance

of T cells anti-tumor cytotoxicity and the consequent reduction in tumor mass. Although the

expression of PD-L1 is mainly attributed to tumor cells, PD-L1 is also expressed by various

immune cells including lymphocytes, monocytes and granulocytes (Fig. 1A).

We previously showed that the neutrophil anti-tumor activity is context dependent and can be

either manifested or inhibited in different tumor microenvironments (circulation, primary tumor,

pre metastatic and metastatic sites). Specifically, we showed that tumor associated neutrophils



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(TAN) do not exhibit anti-tumor functions whereas circulating neutrophils or neutrophils

associated with the premetastatic niche exhibit high anti-tumor cytotoxicity (Gershkovitz et al.,

2018b). To gain insight into the role PD-L1 plays in regulating neutrophil function in cancer, we

evaluated the expression of PD-L1 on neutrophils isolated from the circulation or the primary

tumor. On average, 15% of circulating Ly6G+ neutrophils and ~40% of TAN were PD-L1pos

(Fig. 1B-C) Notably, only a negligible fraction of neutrophils (~1.8%) isolated from the

premetastatic lung were PD-L1pos (Fig. 1D). As we have shown before, neutrophils may be

divided into Normal and Low Density subsets (NDN and LDN respectively, (Sagiv et al., 2015)).

As LDN were shown to possess pro-tumorigenic properties, we hypothesized that PD-L1

expression on neutrophils may be associated with this specific neutrophil subset. Indeed, while

NDN consists of a small PD-L1pos subset (~2%), a significantly higher proportion of LDN are

PD-L1pos (~15% compare Fig. 1E and 1F). LDN are heterogeneous and consist of both mature

and immature neutrophils. Since immature neutrophils were shown to possess pro-tumor

properties (Sagiv et al., 2015; Yang et al., 2011) we sought to determine whether PD-L1pos

neutrophils exhibit a more immature phenotype. To this end we treated tumor-bearing mice with

a single dose of BrdU and assessed the fraction of BrdU+ neutrophils 24 hrs following BrdU

administration (a time point where only immature cells are BrdU+, (Sagiv et al., 2015)). In the

low-density fraction, 16.6% of the BrdU+ (immature) neutrophils were PD-L1pos compared with

only 5.6% of mature (BrdU-) neutrophils (Fig. 1G). The NDN fraction, which contained a small

subpopulation of immature cells, consisted of a low percentage of PD-L1pos neutrophils (~5%),

with no significant difference between the mature and the immature subpopulations (Fig. 1H).

To gain insight into their functional differences, we isolated PD-L1pos and PD-L1neg

neutrophils (Fig. 2A). We found that oxidative burst (the production of H2O2) was significantly

higher in the PD-L1neg neutrophils (Fig. 2B). In addition, we noticed that PD-L1pos cells contain

a larger proportion of morphologically immature neutrophils (Fig. 2C-D). To test whether PD-

L1neg neutrophils exhibit a more pro-inflammatory phenotype compared with PD-L1pos

neutrophils we used the Zymosan-A induced mouse model of peritonitis. This model simulates

self-resolving acute inflammation where peritoneal neutrophil numbers peak at 3 hours post

Zymosan-A injection and gradually decrease over a period of 72 hours. We have previously

shown that the phenotype of the peritoneal neutrophils changes from pro to anti-inflammatory

towards resolution of the inflammatory response (Sagiv et al., 2015). Accordingly, we used this



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model to assess whether the anti-inflammatory, presumably more suppressive neutrophils,

express higher levels of PD-L1. As expected, the percentage of peritoneal Ly6G+ neutrophils

gradually decreased towards resolution (Fig. 2E). Notably, although the percentage of

neutrophils gradually decreased, the fraction of PD-L1pos neutrophils increased with time (Fig.

2F). These results show that the transition of neutrophil function, from pro-inflammatory to pro-

resolution, is associated with an increase in the proportion of PD-L1pos neutrophils and may

indicate a possible immunosuppressive effect of the PD-L1pos neutrophil subpopulation.

PD-1-expressing tumor cells inhibit neutrophil cytotoxicity via PD-L1/PD-1 axis

The notion that PD-L1 expression on neutrophils promotes pro-tumor features was explored

and established in previous studies (Zhang and Xu, 2017). However, the effect of the PD-L1/PD-

1 axis on neutrophil anti-tumor cytotoxicity was never examined. To determine the direct

consequence of PD-1/PD-L1 axis on neutrophils cytotoxicity, we assessed the expression levels

of PD-1 on both neutrophils and tumor cells. We found that while neutrophils do not express PD-

1 (Fig. S1), a significant subpopulation of both 4T1 and AT3 breast cancer cell lines do express

PD-1 (Fig. 3A-B). Importantly, we found that in a co-culture setting, specific blocking of PD-L1

enhanced the susceptibility of both 4T1 and AT3 cells to neutrophil cytotoxicity (Fig. 3C-D).

This observation suggests that PD-1 expression on tumor cells can inhibit neutrophil cytotoxicity

via PD-L1/PD-1 axis. However, this experimental platform suffers from two significant flaws:

First, the PD-L1 antibody can directly affect PD-L1 expressing tumor cells. Second, the addition

of an antibody to the culture may result in neutrophil killing of tumor cells via antibody-

dependent cellular cytotoxicity (ADCC). To overcome these difficulties, we isolated a pure

population of PD-L1neg neutrophils and tested their cytotoxic capacity. Using this approach, we

were able to simultaneously eliminate possible PD-L1/PD-1 interactions andavoid the presence

of an antibody in the culture. We found that when co-cultured with tumor cells, PD-L1neg

neutrophils exhibit a significantly higher cytotoxic ability compared with the mixed population

(Fig. 3E-F) independently corroborating the results obtained by PD-L1 blocking antibody.

Finally, we used PD-1 specific shRNA to knockdown PD-1 expression in both 4T1 and AT3

cells (Fig. 3G-H). We then cocultured control and PD-1kd cells with cytotoxic neutrophils and

found that PD-1kd cells are more susceptible to neutrophil cytotoxicity (Fig. 3I-J). Taken



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together, our observations suggest that tumor cell PD-1 limits neutrophil cytotoxicity and that

PD-L1neg neutrophils have enhanced cytotoxic potential.

Tumor cell PD-1 inhibits neutrophil cytotoxicity and increases metastatic potential

To gain insight into the consequences of PD-1 expression on neutrophil function in the

context of cancer we tested how tumor growth and metastatic progression are affected by

overexpression of PD-1 (Fig. 4A). Notably, PD-1 plays a critical role in mediating the

interaction between tumor cells and lymphocytes which may interfere with proper interpretation

of the experiments. To overcome this difficulty we assessed tumor growth and metastatic

progression in orthotopically (mammary fat pad) injected control vs. PD-1 overexpressing 4T1

tumor cells in NOD-SCID mice. Our data show that PD-1 overexpressing 4T1 tumors exhibited

a more aggressive phenotype as they grew faster (Fig. 4B) and had a significantly higher

metastatic load (Fig. 4C-D) compared to control tumors. Since these mice lack adaptive

immunity, these observations highlight the inhibitory effect of the PD-L1/PD-1 axis on innate

immune cells. However, since this effect on tumor growth and metastatic progression may be

mediated by cells other than neutrophils, we tested the consequences of neutrophil depletion on

metastatic seeding of control and PD-1 overexpressing tumor cells. First, we orthotopically

injected NOD-SCID mice with control 4T1 tumor cells in order to generate a primary tumor and

prime the immune system. Next, we injected control or PD-1 overexpressing 4T1GFP+ tumor cells

via the tail vein to control or neutrophil-depleted tumor-bearing mice (Fig. 4E). Corroborating

previous observations, neutrophil depletion (Fig. 4F) enhanced the seeding of control 4T1GFP+

tumor cells in the lungs (Fig. 4G-H). In contrast, neutrophil depletion had no significant effect

on the seeding of PD-1 over-expressing 4T1GFP+ tumor cells (Fig. 4G and 4I). To broaden the

scope of these observations, we implemented the same approach and overexpressed PD-1 in AT3

cells (Fig. 4J). As in 4T1 cells, neutrophil depletion increased the seeding of control tumor cells

in the lungs (Fig. 4K) but had no effect on metastatic seeding of PD-1 overexpressing cells (Fig.

4K). These observations suggest that PD-1 expression on tumor cells can alter neutrophil

function and inhibit their anti-tumor cytotoxicity.



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DISCUSSION

In recent years our understanding of the multifaceted roles neutrophils play in the tumor

microenvironment is growing, and they may either act to promote or limit tumor growth and

progression. The present study identifies an important role of the PD-L1/PD-1 axis in regulating

the direct anti-tumor function of neutrophils. The PD-L1/PD-1 axis is extensively explored in the

context of cancer where tumor cell PD-L1 interacts with T cell PD-1 to limit adaptive anti tumor

immune responses (Fig. 5A). However, in line with previous studies (He et al., 2015; Wang et

al., 2017), we identified a PD-L1pos subpopulation of neutrophils that is propagated in breast

cancer. Thus far, the role of neutrophil PD-L1 was mainly reported in regard to the suppression

of cytotoxic T cells (Fig. 5B (He et al., 2015)). Moreover, PD-L1pos neutrophils were shown to

contribute to the growth and progression in human tumors and are considered to be a poor

prognostic factor indicative of reduced patient survival (Wang et al., 2017). In line with these

studies, we observed a high expression of PD-L1 on the non-cytotoxic tumor infiltrating

neutrophils (Fig. 1C). In contrast, cytotoxic neutrophils isolated from the circulation or the pre-

metastatic niche are relatively low in PD-L1 expression (Fig. 1B and 1D). PD-L1pos neutrophils

exhibit a pro-tumor phenotype and can be mainly classified as immature LDN (Fig. 1G). This

observation provides further support and possible mechanistic insight into the

immunosuppressive LDN phenotype (Sagiv et al., 2015). Taken together, these observations

suggest that PD-L1pos neutrophils are characterized by having an immature, low-density, pro-

tumor phenotype.

The role of PD-L1 expressing neutrophils in the context of T cell suppression and tumor

growth was already explored (Fig. 5B). However, the role played by the PD-L1/PD-1 axis in

regulating anti-tumor properties of neutrophils was unknown. We and others (Kleffel et al.,

2015) have shown that a subpopulation of tumor cells express PD-1 which may facilitate

interactions with PD-L1 expressed by other cells. Accordingly we hypothesized that tumor cell

PD-1 may interact with neutrophil PD-L1 and thus modulate neutrophil function. Indeed,

blocking of the PD-L1/PD-1 axis in a co-culture setting enhanced tumor cell susceptibility to

neutrophil cytotoxicity suggesting that the PD-1/PD-L1 axis is involved in regulating the direct

neutrophil/tumor cell crosstalk. Moreover, this observation indicates that the PD-1/PD-L1

interaction in this context reduces the extent of neutrophil cytotoxicity (Fig. 5C). Since PD-1

expression levels vary among different tumors, we speculated that the effect different tumors



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have on the function of PD-L1pos may correlate with the extent of PD-1 expression. To test this

idea we overexpressed PD-1 in tumor cells and assessed tumor growth and progression mouse

models of breast cancer. This led to a more aggressive phenotype as the tumors grew faster and

had a higher metastatic load. Furthermore, neutrophil depletion dramatically enhanced metastatic

seeding of control cells whereas PD-1 over-expressing tumor cells were not affected. Together,

the observations implicate neutrophils in this process and suggest that PD-1 overexpressing cells

are protected from neutrophils. Importantly, these experiments were done using immunodeficient

NOD-SCID mice to eliminate the contribution of lymphocytes.

In summary, this study provides novel insight into the interaction of neutrophil PD-L1 with

tumor cell PD-1 and the role it plays in regulating neutrophil cytotoxicity. We show that the PD-

L1/PD-1 axis is also critical for the direct cytotoxicity,emphasizing the beneficial effect of

blocking the PD-L1/PD-1 axis in cancer not only to enhance T cells functions, but also to

enhance neutrophil anti-tumor activity.

FIGURE LEGENDS

Figure 1. PD-L1pos neutrophils are associated with immature, low-density phenotype.

A. FACS analysis of PD-L1pos cells (red) in whole blood from a tumor-bearing mouse. B-D.

FACS analysis of PD-L1 expression in Ly6G+ neutrophils isolated from the circulation (B), the

primary tumor (C) and the premetastatic lung (D) of a tumor-bearing mouse. Red gate represents

the PD-L1pos subpopulation. E-F. FACS analysis of PD-L1 expression in Ly6G+ neutrophils

normal-density neutrophils (E) and low-density neutrophils (F) from a tumor-bearing mouse, red

gate represents PD-L1pos subpopulation. G-H. FACS analysis of BrdU staining in circulating

Ly6G+ low-density neutrophils (G) and normal-density neutrophils (H) from a 4T1 tumor-

bearing mouse. Red gate represents PD-L1pos subpopulation.

Figure 2. PD-L1pos neutrophils are associated with the resolution phase in inflammation

A. FACS analysis PD-L1 expression in presorted neutrophils (Pre), PD-L1neg neutrophils (red)

and PD-L1pos neutrophils (green). B. Spontaneous ROS production in PD-L1neg (red) and PD-

L1pos (green) neutrophils. C. Percent of immature cells in PD-L1neg (red) and PD-L1pos (green)

neutrophil population. D. Representative images of H&E staining of PD-L1pos and PD-L1neg

neutrophils. E. Time dependent changes in the fraction of peritoneal neutrophils (of all CD45+



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cells) in self-resolving peritonitis. F. Time dependent changes in the fraction of peritoneal of PD-

L1pos neutrophils (of all Ly6G+ cells) in self-resolving peritonitis.

Figure 3. Blocking the PD-1/PD-L1 axis increases tumor cell susceptibility to neutrophil

cytotoxicity

A. FACS analysis of PD-1 expression in 4T1 breast tumor cells. B. FACS analysis of PD-1

expression in AT3 tumor cells. C-D. Neutrophil mediated killing of 4T1 (C) and AT3 (D) tumor

cells in the absence (cont.) or presence of PD-L1 blocking antibody. E-F. Neutrophil mediated

killing of 4T1 (E) and AT3 (F) tumor cells with unsorted neutrophils (cont.) or PD-L1neg

neutrophils G-H. FACS analysis of PD1 expression in PD-1kd 4T1 (G) and AT3 (H) cells tumor

(red). I-J. Neutrophil mediated killing of control and PD-1kd 4T1 (I) and AT3 (J) tumor cells.

*p<0.05, **p<0.01.

Figure 4. PD-1 overexpression impairs neutrophil cytotoxicity in NOD-SCID mice.

A. FACS analysis of PD-1 overexpression in 4T1 tumor cells. B. Tumor growth of orthotopically

injected control and PD-1 overexpressing 4T1 cells (n=5). C. Representative H&E staining of

lungs from mice injected with control or PD-1 overexpressing 4T1 cells (n=5). D. Total number

of spontaneous metastases in control and PD-1 overexpressing 4T1 tumor bearing mice (n=5). E.

Experimental metastatic seeding assay - female NOD-SCID mice were orthotopically (mammary

fat pad) injected with control (4T1 or AT3) tumor cells. Starting on day 6 the mice were injected

daily (arrows) with either control IgG or neutrophil depleting anti-Ly6G antibody i.p. On day 8

the mice were injected with 5x105 GFP+ control or PD-1 overexpressing cells (4T1 or AT3) via

the tail vein. On day 10 the number of lung associated GFP+ cells were analyzed by fluorescent

microscopy. F. FACS analysis of circulating Ly6G+ neutrophils in control and neutrophil

depleted tumor-bearing mice. G. Average number of lung-associated GFP+ control and PD-1

overexpressing 4T1 tumor cells in control and neutrophil depleted mice (n=5). H. Representative

images of lung associated control GFP+ cells in control mice (control) and neutrophil depleted

mice (control + dep.). I. Representative images of lung associated PD-1 overexpressing GFP+

cells in control mice (PD-1 o.e.) and neutrophil depleted mice (PD-1 o.e. + dep.). J. FACS

analysis of PD-1 overexpression in AT3 tumor cells. K. Average number of lung-associated



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GFP+ control and PD-1 overexpressing AT3 tumor cells in control and neutrophil depleted mice

(n=5). *p<0.05, **p<0.01.

Figure 5. Mechanisms of tumor immune evasion mediated by the PD-1/PD-L1 axis

A. Tumor cell expressed PD-L1 serves as a ligand for PD-1 expressed on cytotoxic T Cells. This

interaction leads to blocking of anti tumor T cell responses. Targeting this interaction serves as

the basis for anti cancer immunotherapy. B. Neutrophils possessing immunosuppressive

properties express PD-L1 which serves as a ligand for PD-1 expressed on cytotoxic T cells,

blocking T cell anti tumor responses. C. Tumor cell expressed PD-1 interacts with PD-L1

expressed by neutrophils blocking cytotoxic anti tumor neutrophil responses.

MATERIALS AND METHODS

Animals

5-6-week-old BALB/c and NOD-SCID mice were purchased from the Harlan (Israel). All

experiments involving animals were approved by the Hebrew University’s Institutional Animal

Care and Use Committee (IACUC).

Cell Lines

4T1 and AT3 mouse breast cancer cells were purchased from the ATCC and cultured in DMEM

containing 10% heat-inactivated fetal calf serum (FCS). 4T1 and AT3 cells were transduced with

a lentiviral vector (pLVX-Luc, MigR1-Luc or the triple-modality reporter gene TGL (Minn et

al., 2005)) to stably express firefly luciferase. PD1kd 4T1 and AT3 cells were generated by

lentiviral transduction with PD1 specific shRNAs from Sigma (TRCN0000097670-74). Control

cells were transduced an empty vector (pLKO). For killing assay neutrophils and tumor cells

were cultured in RPMI containing 2% heat-inactivated fetal calf serum (FCS).

Plasmids

Retroviral MigR1-Luc vector: The open reading frame of Firefly luciferase was prepared from

pLVX-luciferase using forward primer 5'-CAG TCC GCT CGA GGC CGC CAC CAT GGA

AGA CGC CAA AAA CAT AAA G-3' containing a XhoI restriction site, Kozac sequence



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followed by the ATG initiation code and reverse primer 5'-ACT TCC GGA ATT CTT ACA

CGG CGA TCT TTC CGC CC-3' containing an EcoRI restriction site and the stop codon and

Phusion Flash High-Fidelity PCR master mix (Thermo Scientific). The amplified Luciferase

PCR product was digested by XhoI and EcoRI and inserted into the XhoI/EcoRI site of the

MigR1 plasmid.

Mouse PD1 expression vector: Mouse PD1 was prepared by PCR on cDNA from 4T1 breast

cancer cells using Phusion Flash High-Fidelity PCR master mix (Thermo Scientific) and the

following primer pair: F' ACT TCC GGA ATT CGC CGC CAC CAT GTG GGT CCG GCA

GGT ACC CTG GTC containing a EcoRI site and nt 67-89 of Pdcd1 (NM_008798.2) and R'

AAGGAAAAAAGCGGCCGCTCAAAGAGGCCAAGAACAATGTCC containing a NotI site

and nt 907-930 of Pdcd1. The EcoRI/NotI-cut PCR product was mixed with annealed Flagx3

primers having sticky ends of EcoRI and NotI: F: ACT TCC GGA ATT CGC CGC CAC CAT

GTG GGT CCG GCA GGT ACC CTG GTC and R' AAG GAA AAA AGC GGC CGC TCA

AAG AGG CCA AGA ACA ATG TCC; and inserted into the EcoRI/NotI site of the pLVX-Puro

vector. The plasmid was sequenced at the Center for Genomic Technologies at the Givat Ram

Campus, The Hebrew University of Jerusalem.

Neutrophil Purification

Neutrophils were purified from orthotopically injected (mammary fat pad) 3-week 4T1 tumor-

bearing mice as described before (Sionov et al., 2015a). In brief, whole blood was collected by

cardiac puncture using heparinized (Sigma) syringe. The blood was diluted with 5 volume of

PBS containing 0.5% BSA and subjected to a discontinuous Histopaque (Sigma) gradient (1.077

and 1.119). NDN were collected from the 1.077-1.119 interface. LDN were collected from the

plasma-1.077 interface. Red blood cells (RBCs) were eliminated by hypotonic lysis. Neutrophil

purity and viability were determined visually and were consistently >98% for NDN.

Isolation of low and normal density neutrophils

Circulating NDN and LDN were purified from 4T1 tumor bearing mice using Histopaque

gradient.



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Primary tumor and metastasis formation in vivo assay

NOD-SCID mice were injected into the mammary fat pad with 1x106 control or PD1 over-

expressing 4T1 cells. Tumors were measured weekly. On day 27 post tumor engraftment the

mice were sacrificed and the lungs were excised for analysis metastatic spread. The lungs were

sectioned with 100 µm intervals and stained with H&E. The number of metastatic foci was

determined by histological examination. Data represents the average tumor size of at least 5 mice

per group.

Metastatic seeding assay

4T1 metastatic seeding assay: Female NOD-SCID mice were orthotopically (mammary fat pad)

injected with parental 4T1 tumor cells. On day 6 the mice were randomized and injected daily

with either control IgG or neutrophil depleting anti-Ly6G (50 µg/mouse i.p.). On day 8 the mice

were injected to the tail vein with 5x105 GFP+ cells. On day 10 the lung associated GFP+ cells

were analyzed by fluorescent microscopy.

AT3 metastatic seeding assay: Female NOD-SCID mice were orthotopically (mammary fat pad)

injected with parental 4T1 tumor cells. On day 14 the mice were randomized and injected daily

with either control IgG or neutrophil depleting anti-Ly6G (50 µg/mouse i.p.). On day 16 the

mice were injected to the tail vein with 5x105 GFP+ cells. On day 18 the lung associated GFP+

cells were analyzed by fluorescent microscopy.

qPCR

Total RNA was isolated with TRI-Reagent (Sigma) according to the manufacturer’s instructions.

RNA was reverse transcribed into cDNA using the AB high capacity cDNA kit (Applied

Biosystems). 2 ng of converted cDNA was used for each reaction, using Kapa Sybr-Green

Master Mix (Kapa Biosystems) and 300 nM of forward/reverse primer set. Analyses were done

using the CFX384 BioRad Real Time PCR. GAPDH was used as a reference gene. The

following primer sequences were used for gene expression analyses: GAPDH-F 5'-GCC TTC

CGT GTT CCT ACC-3', GAPDH-R 5'-CCT GCT TCA CCA CCT TCT T-3',



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Peritonitis

BALB/c mice were injected i.p. with 1mg of Zymosan A from Saccharomyces

cerevisiae (Sigma) diluted in 1ml of sterile PBS. Cells were retrieved using peritoneal lavage at

four different time points (3, 24, 48, and 72 hours post i.p. injection). After purification and red

blood cells (RBCs) hypotonic lysis the cell pellet was stained for PD-L1 and Ly6G and measured

by flow cytometry.

ROS assay

Purified PD-L1pos and PD-L1neg neutrophils were plated (2x105) in 180 µl of HBSS (Biological

Industries) in white 96-flat bottom wells (Corning) containing 50µM Luminol (Sigma). ROS

production (chemiluminescence) and was measured using Tecan F200 microplate luminescence

reader.

In Vitro Killing Assay

Luciferase-labeled tumor cells (1x104/well) were plated in 100 µl RPMI-1640 with 2% FCS in

white 96-flat bottom wells (Corning). After 24 hours incubation, purified neutrophils (1x105/well

in 100 µl) were added to the plated tumor cells and co-cultured overnight in the presence or

absence of anti-PD-L1 2µg (BioLegend). Following overnight incubation, luciferase activity was

measured using Tecan F200 microplate luminescence reader. Extent of killing was determined

by the ratio between tumor cells cultured alone and tumor cells co-cultured with neutrophils. In

vitro experiments were repeated at least three times.

PD-L1 positive and negative neutrophil isolation

PD-L1 positive and negative neutrophils were isolated using EasySepTM PE positive selection kit

(STEMCELL) according to the manufacturer’s instructions.

BrdU labeling

Tumor bearing mice were injected i.p. with 100µL of BrdU solution (10mg/mL in sterile PBS,

BD Pharmingen). Incorporation of BrdU was detected using the FITC BrdU Flow Kit (BD

Pharmingen) and analyzed by flow cytometry.



https://doi.org/10.1101/2020.02.28.969410


Antibodies

Mouse α-PD-L1 (BioLegend), α-mouse PD-L1-PE (BioLegend), α-mouse PD1-APC

(BioLegend), α-mouse Ly6G-VioBlue (TONBO Biosciences), α-mouse Ly6G (clone 1A8,

BioXCell).

Statistical Analysis

For studies comparing differences between two groups, we used unpaired Student’s t tests.

Differences were considered significant when p < 0.05. Data are presented as mean ± SEM.

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1.81%

-101 102 1030

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PD-L1103-102 104 105

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https://doi.org/10.1101/2020.02.28.969410


PD-1 PD-L1

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Figure 5



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the pd-l1/pd-1 axis blocks neutrophil cytotoxicity in cancer · 28/02/2020 · study (granot et...

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